The Nef protein of HIV-1 is essential to infectivity by virtue of its ability to undermine host cellular defenses. Nef has many activities, but the most significant is thought to be the downregulation of CD4. CD4 is the primary cell surface receptor for HIV-1. The sustained presence of CD4 on the cell surface during infection interferes with Env incorporation into virions and with the release of new infectious particles. Nef internalizes CD4 by recruiting it to clathrin-coated pits. Nef binds directly both to the cytosolic tail of CD4 and to the AP-2 clathrin adaptor protein, thereby serving as a double-headed bridge between the two. Nef also prevents immune recognition of infected cells by sorting MHC-I not to the plasma membrane, but to lysosomes, where it is degraded. Nef accomplishes this by directly bridging CD4 and MHC-I to the clathrin adaptor complexes AP-1 and AP-2. Our laboratory solved two structures in 2013 and 2014 that revealed that the Nef binding site only becomes exposed when AP complexes are unlocked. In this proposal, we explore the novel hypothesis that Nef is not only a bridge from cargo to AP adaptor, but also an allosteric activator of AP complexes. If verified, an allosteric role could open new avenues for the targeting of Nef by antiretrovirals. This is excitin because it is significantly easier to target allosteric transitions with small molecules as compare to protein:protein interfaces. The project will integrate the three most powerful modalities of structural and biophysical research: cryoelectron microscopy, single molecule spectroscopy, and x-ray crystallography. The specific aims of the project are as follows. 1. We will determine whether and how HIV-1 Nef acts alone or together with Arf1 to allosterically activate AP-1. We will isolate stable, activated complexes of AP-1, Arf1, Nef, and cargo. The structures of the complexes will be determined by cryoEM alone and as bound to membranes. 2. In order to have a direct probe for the conformation of AP-1 on membranes that is orthogonal to activator and cargo binding, we will create fluorescent reporters for the dynamics, diffusion, and conformation of AP-1. Single particle tracking, bulk FRET, and single molecule FRET will be employed in this aim to determine how many points of contact activated AP-1 has with membranes, what its conformation is, and to discover the nature and lifetime of the activating conformational change. 3. The project will culminate in the characterization and structure determination of complexes of activated AP-1 and AP-2 with Nef and the CD4 cytosolic tail. Structures will be determined in parallel by cryoelectron microscopy and x-ray crystallography. This will lead to the discovery of the interfaces involved in molecular recognition at each stage in activation and CD4 binding. The function of interfaces will be dissected through in vitro assays of binding, activation, and dynamics, and cell-based assays of CD4 internalization and lysosomal sorting.